Biochemistry 1985, 24, 7273-1278
Lowry, 0. H., Rosebrough, N. J., Farr, A. L., & Randall, P. (1951) J . Biol. Chem. 193, 265-274. Morris, S. J., Schultens, H. A., & Schober, R. (1977) Biophys. J . 20, 33-48. Njus, D., & Radda, G. K. (1978) Biochim. Biophys. Acta 463, 2 19-244. Njus, D., & Radda, G. K. (1979) Biochem. J . 180,579-585. Niedermaier, W., & Burger, A. (1981) Naunyn-Schmiedeberg's Arch. Pharmacol. 316, 69-80. Phillips, J. H. (1981) Biochem. J . 200, 99-107.
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Phillips, J. H., Allison, Y . P., & Morris, S. J . (1977) Neuroscience (Oxford) 2, 147-152. Sen, R., & Sharp, R. R. (1982) Biochim. Biophys. Acta 721, 70-82. Sillen, L. G., & Martell, A. E. (1971) Spec. Pub1.-R. SOC. Chem. (Suppl. I ) No. 25, 623. Smith, A. D., & Winkler, H. (1967) Biochem. J . 103, 483-492. Trifaro, J. M., & Dworkind, J. (1970) Anal. Biochem. 34, 403-412.
Mutagenesis by N4-Aminocytidine: Induction of AT to GC Transition and Its Molecular Mechanism? Kazuo Negishi, Mitsuko Takahashi, Yasuhiro Yamashita, Masahiko Nishizawa, and Hikoya Hayatsu* Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan Received May 15, 1985
a potent mutagen toward Escherichia coli and Salmonella typhimurium. It induced reversion of an amber mutant of 4 x 1 7 4 phage (am3) to the wild type. This reversion was shown to be exclusively due to the A T to GC transition. It is likely that N4-aminocytidine is metabolized within the bacterial cells into N4-aminodeoxycytidine 5'-triphosphate and this nucleotide is incorporated into D N A during the multiplication of the cells and the phages, thereby causing base-pair transitions. The molecular basis for this erroneous replication was obtained in studies of in vitro incorporation of N4-aminodeoxycytidine 5'-triphosphate into polynucleotides catalyzed by the E. coli D N A polymerase I large fragment. The results have shown that this cytosine analogue can be efficiently incorporated as a substitute of cytosine and that it can also be incorporated as a substitute of thymine. The ratio in the rate of the N'-aminocytosine nucleotide incorporation to that of natural nucleotide incorporation was 1/2 to cytosine and 1/30 to thymine. Furthermore, the N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite to them. ABSTRACT: N4-Aminocytidine is
R e c e n t l y , we have found that N4-aminocytidine can efficiently induce base-change mutations in bacteria and in phages (Negishi et al., 1983). Its high activity is exceptional for a nucleoside analogue and is comparable to that of N-methylN'-nitro-N-nitrosoguanidine, one of the most potent mutagens. The activity is more than 1000 times stronger than that of 2-aminopurine in the reversion of Salmonella typhimurium TAlOO as well as in the reversion of Escherichia coli WP2 trp (ocher). Thus, the numbers of revertants formed per nanomole of reagent (calculated from the linear dose-responses) were, N4-aminocytidine/2-aminopurine/N-methylN'-nitro-N-nitrosoguanidine, in S . typhimurium TAlOO 601 0.02/300 and in E . coli WP2 uvr 160/0.012/10 (Negishi et al., 1983; unpublished work). Probably, within the cells N4-aminocytidine is converted to dCamTP'and incorporated into DNA. The principal tautomeric structure of N4-aminocytosine is the amino form (Brown et al., 1968; Takayanagi et al., 1980), and Brown et al. (1968) have estimated that about of the molecules is present in the imino form. A simple mechanism for the N4-aminocytosine-induced mutagenesis would be that the imino form of N"-aminocytosine pairs with adenine, while the major amino form pairs with guanine (Figure 1). Thus, during one rep?This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
0006-2960/85/0424-7273$01.50/0
licational cycle, dCamTPis incorporated into DNA mostly as a substitute of dCTP, but it can also be incorporated to a certain extent as a substitute of dTTP (see step 1 in Figure 1). In the next cycle of replication, either dGTP or dATP can be incorporated opposite N4-aminocytosine on the template DNA (step 2). This scheme is analogous to the classical one proposed by Freese (1959) for the base analogue mutagenesis. The mechanism in Figure 1 predicts that N4-aminocytidine should induce both the AT to GC and the GC to AT transitions and that no transversions should be caused. In the present work, we analyzed the mutational specificity in the N4-aminocytidine-inducedreversion of $X 174 am3 phage and studied the in vitro utilization of dCamTPin the polynucleotide synthesis catalyzed by E . coli DNA polymerase I large fragment. The results obtained by sequencing the revertant phage DNAs have shown that a AT to GC transition took place in the am3 site. Furthermore, the study on the in vitro DNA synthesis gave strong support for the mechanism as illustrated in Figure 1.
'
Abbreviations: dCamTP,N4-amino-2'-deoxycytidine 5'-triphosphate; dCohTP,N4-hydroxy-2'-deoxycytidine 5'-triphosphate; imdCamTP(and imdCamMP), 6-(2'-deoxy-~-~-ribofuranosyl)-5-oxo-5,6-dihydro-~-triazolo[4,3-c]pyrimidine 5'-triphosphate (and 5'-monophosphate); HPLC, high-pressure liquid chromatography; HEPES, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid.
0 1985 American Chemical Society
B I oc H E M I S T R Y
7274
Ambiguous base pairing
N E G I S H I ET A L . '
Cam G (amino form)
Cam
A
( imino form)
Mutagenes/s Step 1
Pathway 1
-T -
.
-A-
dCamTP
-
S'ep2
-cam
.
-
-A-
4
- Cam -A -
-cam-G -
[AT
-
GC)
- Cam Pathway2
-C
-
-G
.-
d c a m ~ ~-Cam
-
- G .-
,T
-A -
(GC -AT % -cam-
]
-G-
q, major event ; FIGURE
-,
minor event
1: Mechanism of N4-aminocytidine-inducedbase-pair tran-
sitions. AND METHODS MATERIALS Materials. Poly(dA), oligo(dT)12-18,oligo(dA)12-18,oligo(dG)12-1S,and the copolymers with alternating AT or GC sequences poly(dA-dT) and poly(dG-dC) were products of P-L Biochemicals. Activated calf thymus DNA was prepared according to Aposhian & Kornberg (1962). E . coli DNA polymerase I large fragment, terminal nucleotidyl transferase, and restriction endonucleases were obtained from Takara Shuzo. Radioactive chemicals were products of Amersham. dCohTPwas synthesized from dCTP by treatment with hydroxylamine (Budowsky et al., 1972) and purified with HPLC. Analysis of 4x174 Phage Revertants. 4x174 phage am3 was mutagenized by allowing the phage-infected E . coli HF47 14 (sup') to be cultured in a medium containing 0.05 mM N4-aminocytidine (Negishi et al., 1983). The revertants were detected by the growth on E. coli CI (sup-). Among the revertants obtained, eight colonies were randomly selected. They were obtained in three independent experiments. Each colony was cultured to obtain RFI DNA (covalently closed circular replicative form) by the method of Ueda et al. (1981). The RFI DNA was digested with restriction endonuclease TaqI, and the DNA fragment containing am3 site (231 base pairs) was separated from other fragments by polyacrylamide gel electrophoresis. The fragment was further digested with HaeIII, and a 179 base pair piece containing am3 site was isolated. The DNA fragment was sequenced by the MaxamGilbert (1977) procedure (see Figure 2). Only about a 60nucleotide region encompassing the am3 site was sequenced. Preparation of N4-Aminocytosine Derivatives. N4Aminocytidine was prepared from cytidine as described (Negishi et al., 1983). dCamTPand dCamMPwere similarly obtained from dCTP and dCMP, respectively. Thus, a solution containing 0.1 M dCTP, 4 M hydrazine, and 0.1 M sodium bisulfite at pH 7 (the pH was adjusted by addition of HCl) was incubated at 60 OC for 4 h. The reaction mixture was fractionated by HPLC on a column of Partisil lOSAX (4.6 X 250 mm) (1 5-pL reaction mixture for a run). The column was eluted with 0.3 M ammonium phosphate at pH 3.8 at a flow rate of 2 mL/min. The peak fraction detected by A254 was pooled and applied to a column of LiChrosorb RP18 (4.6 X 250 mm). The column was eluted with 10 mM triethylammonium bicarbonate-3% methanol at pH 6.3 at a flow rate of 1 mL/min. The dCamTPfraction obtained was lyophilized and stored at -80 "C. [3H]dCamTPwas prepared similarly
from [ 1',2',5-3H]dCTP. In this case, nonradioactive dCamTP had been added as a carrier at the start of the second HPLC fractionation. Specific radioactivity of the [3H]dCamTPprepared was 500 Ci/mol. Purity of the dCamTPwas examined by derivatization with ethyl acetimidate to form the triazolopyrimidine compound imdCamTP (Nitta et al., 1984; N. Nitta and H. Hayatsu, unpublished work). This was necessary because dCamTPgave a tailing peak in the HPLC, a part of which overlapping with the peak for dCTP. HPLC analysis of the imdCamTPon Partisil lOSAX with an eluting solvent, 0.3 M ammonium phosphate (pH 3.8)-acetonitrile, lo/ 1, showed that the [3H]dCamTPsample was more than 98.8% pure (contents of contaminating [3H]dCTP and [3H]dUTP were
